The biosynthesis of the starch granule is a complex process which involves the action of an array of isoforms of enzymes involved in the starch biosynthesis. Following the formation of glucose-1-phosphate, the enzyme activities required for the synthesis of granular starch include ADP glucose pyrophosphorylase (EC 2.7.7.27), starch synthases (EC 2.4.1.21), branching enzymes (EC 2.4.1.18) and debranching enzymes (EC 3.2.1.41 and EC 3.2.1.68) (Mouille et al., 1996). Plants contain isozymes of each of these activities, and the definition of these isoforms and their roles has been conducted through investigation of the properties of the suite of soluble enzymes found in the stroma of the plastid, analysis of the proteins entrapped within the matrix of the starch granule, and mutational studies to identify genes and define linkages between individual genes and their specific roles.
Starch synthases extend regions of α-1,4 glucan through the transfer of the glucosyl moiety of ADPglucose to the non-reducing end of a pre-existing α-1,4 glucan. In addition to GBSS, 3 other classes of starch synthase have been identified in plants, SSI (wheat, Li et al., 1999 and GenBank Accession No. U48227; rice, Baba et al., 1993; potato, Genbank Accession No.-Y10416, SSII (pea, Dry et al. 1992; potato, Edwards et al., 1995; maize, Harn et al., 1998 and GenBank Accession No. U66377) and SSIII (potato, Abel et al., 1996; maize, Gao et al., 1998. In the cereals, the most comprehensively studied species is maize, where in addition to GBSS, cDNAs encoding SSI, and SSIIb have been isolated, and both cDNA and genomic clones for dull1 have been characterised (Knight et al., 1998; Harn et al., 1998; Goa et al., 1998). In maize, the product of the du1 gene is known as maize SSII, however this gene is the homologue of potato SSIII.
The proteins within the matrix of the wheat starch granule have been extensively studied (Denyer et al., 1995; Rahman et al., 1995; Takaoka et al., 1997; Yamamori and Endo, 1996) and 60, 75, 85, 100, 104 and 105 kDa protein bands can be visualised following SOS-PAGE. The predominant 60 kDa protein is exclusively granule-bound and is analogous to the ‘waxy’ granule bound starch synthase (GBSS) gene in maize (Rahman et al., 1995). The combination of three null alleles for this enzyme from each of the wheat genomes (Nakamura et al., 1995) results in the amylose-free ‘waxy’ phenotype found in other species The 75 kDa starch synthase I (wSSI) is found in both the granule and the soluble fraction of wheat endosperm (Denyer et al., 1995; Li et al., 1999) and has been assigned to chromosomes 7A, 7B and 7D (Yamamori and Endo, 1996; Li et al., 1999). The 85 kDa band contains a class II branching enzyme and an unidentified polypeptide (Rahman et al., 1995). The 100, 104 and 105 kDa proteins of the wheat starch granule (designated Sgp-B1, Sgp-D1 and Sgp-A1 by Yamamori and Endo, 1996) have been shown to be encoded by a homeologous set of genes on the short arm of chromosome 7B, 7A and 7D respectively (Yamamori and Endo, 1996; Takaoka et al., 1997). Denyer et al. (1995) concluded on the basis of enzyme activity assays that these proteins were also starch synthases. These genes are referred to hereinafter as the “wheat SSII genes”.
While GBSS has been established to be essential for amylose synthesis, the remaining starch synthases are thought to be primarily responsible for the elongation of amylopectin chains, although this does not preclude them from also having non-essential roles in amylose biosynthesis. Differences in kinetic properties between isoforms, and the analysis of mutants lacking various isoforms, suggests that each isoenzyme contributes to the extension of specific subsets of the available non-reducing ends.